In recent years, the development of genetic engineering techniques has had dramatic implications in the field of crop improvement. Using these techniques, beneficial traits can be introduced into almost any crop and improved crops can be rapidly obtained. The use of genetic engineering obviates the need for lengthy procedures to introduce the desired trait by conventional breeding methods.
Present plant transformation methods generally integrate a single transgene into the host genome. Successful integration of each transgene requires repeated confrontation of various issues, such as variability in transgene expression caused by different integration loci, so-called “positions effects,” and the risk of creating a mutation in the genome upon integration of the transgene into the host. Consequently, a large number of transformation events must be screened and tested before obtaining a transgenic plant that exhibits the desired level of transgene expression without also exhibiting abnormalities resulting from the inadvertent insertion of the transgene into an important locus in the host genome. Moreover, if an additional transgene is subsequently added to a transgenic plant, the additional transgene likely will be integrated into the genome at a location that is different from the location of the pre-existing transgene, rendering the breeding of elite plant lines with both genes difficult and cumbersome.
An inherent problem with such single-round integration techniques is that sequence stacking, or the successive integration of multiple nucleotide sequences at a predetermined locus in the host genome, is difficult to accomplish. However, efficient sequence stacking is desirable for a variety of reasons. For example, the ability to achieve targeted insertion of multiple transgenes into a host would facilitate registration of a transgenic plant with government regulatory agencies, since the potential for random alteration of the plant's genetic material would be minimized. Further, in some cases, such as the engineering of traits or metabolic pathways that involve multiple genes, for example, co-location of the transgenes would be highly desirable. Additionally, since only a limited number of selectable and scoreable marker sequences may be available for use in transforming a given crop, the ability to re-use a marker sequence when introducing successive nucleotide sequences into the host genome would also be desirable.